MicroRNAs (miRNAs) comprise a newly discovered class of non-coding RNA genes and specific miRNAs have already been linked to key gene regulatory roles relevant to human health and disease. The let-7 gene is a founding member of this novel family of regulatory molecules and it serves as a model for understanding the molecular mechanisms that control miRNA biogenesis and function. Furthermore, let-7 is exceptionally well conserved across species and its potential role as a tumor suppressor in humans is related to its function in promoting cellular differentiation, originally determined in C. elegans. The research described in this renewal builds upon our identification of dynamic processes that regulate biogenesis of let-7 miRNA and our development of a robust biochemical approach aimed at revealing how the miRNA complex, which includes the argonaute protein ALG-1, recognizes and regulates direct target genes in vivo. Our studies are broadly relevant for understanding how miRNAs are expressed and function across species and also how perturbation of these pathways contributes to human disease. To test the hypothesis that specific sequences and proteins cooperate to regulate expression and function of let-7 and related miRNAs, we propose the following aims. 1. Characterize the post-transcriptional mechanisms that control expression of let-7 family miRNAs. (A) The cis-acting elements that regulate processing of let-7 miRNA will be determined using in vitro structure analyses and in vivo expression and function assays. (B) The primary transcripts and elements that mediate post-transcriptional regulation of let-7 related miRNAs (mir-84, mir-48, mir-241 and mir-795) will be defined. 2. Determine the function of the LIN-28 and LIN-42 proteins in regulating the expression of miRNAs. (A) The role of lin-28 in let-7 miRNA biogenesis will be investigated by in vivo and in vitro RNA binding assays. A sensitive RNAi based screen will be undertaken to identify both enhancers and suppressors of lin-28 mutant phenotypes that are dependent on let-7 activity. (B) A novel function for the Period homolog, LIN-42, in regulating miRNA maturation will be investigated by determining RNA targets and genetic interactors. 3. Identify in vivo miRNA target sites bound by ALG-1 and the regulatory outcome of the interactions. (A) Sequences bound and regulated by ALG-1 that are dependent on specific miRNAs will be identified using a novel biochemical and computational approach combined with microarray analyses. (B) Specific miRNA targets will be validated by high throughput genetic approaches and sensitive reporter gene assays. PUBLIC HEALTH RELEVANCE: Normal cells become cancerous when gene regulatory pathways are disrupted. MicroRNAs comprise a new class of gene regulatory molecules, and aberrations in the expression and function of specific microRNAs contribute to oncogenesis. By studying the essential role of microRNAs in normal cellular development, this proposal aims to help elucidate how disruption of this pathway can lead to cancerous states.